The immune system of the gastrointestinal tract protects the host from pathogen invasion, while facilitating colonization of a diverse, symbiotic microbiota. A clear understanding of the mechanisms that regulate this delicate balance is lacking, but specialized secretory epithelial cells, called Paneth cells, appear to be important contributors. Abundant evidence supports that defect in Paneth cell function increases susceptibility to enteric pathogens and to chronic inflammatory bowel disease. Alpha-defensins are the most abundant effector molecules of Paneth cells and contribute to both host defense and homeostasis, but their precise functions and contributions to these processes are not fully known. The objective for this proposal is to develop innovative new mouse models that will serve as valuable tools to delineate the relative magnitude and specific contributions of Paneth cell alpha-defensins to homeostasis and host defense. These models will enable us to test two interrelated hypotheses: (i) Paneth cell alpha-defensins are major contributors to both intestinal homeostasis with the microbiota and host defense against enteric bacterial pathogens, and (ii) the two human -defensins, HD5 and HD6, have non-redundant roles in small intestinal innate immunity. Aim #1 will develop a novel Paneth cell alpha-defensin knockout model that overcomes the limitations of currently available models. Using this model, we will investigate the specific contribution of Paneth cell alpha-defensins on both baseline composition of the colonizing microbiota and susceptibility to enteric pathogens. Aim #2 will elucidate the non-redundant roles HD5 and HD6 in host defense against enteric pathogens through analysis of transgenic mice that express physiological concentrations of these defensins in the absence of endogenous mouse alpha-defensins. By studying Paneth cell alpha-defensin function with new and innovative mouse models, the proposed research will generate insights on intestinal innate mucosal immunity.